The aim of this Project is to develop and utilize oligonucleotide microarray technology for the model filamentous fungus Neurospora crassa. These studies will provide a solid foundation of gene expression profiles for the filamentous fungi that will promote comparative studies within this group and between the fungi and other organisms. The rich foundation of biological and molecular tools available for Neurospora has been complemented with an archival quality genomic sequence, thus creating the opportunity for asking questions of genome regulation on a global scale. Gene expression data generated by microarray technology as described in this proposal can measurably enhance the functional analysis of this organism's genes. Our first objective is to generate slides containing 70-met oligonucleotides representing each of the predicted genes of N. crassa and to use these slides to standardize our techniques to develop sound statistical analysis of microarray data. Our second objective is to carry out base-line transcriptional profiling on the sequenced strain of Neurospora following exposure to a variety of growth conditions and environmental stresses. Our third objective will test hypotheses derived from microarray experiments using knockout strains generated from Project #1 of this proposal. Anticipated knockouts will include (1) genes that have been annotated in N. crassa that are involved in transcriptional regulatory processes in S. cerevisiae and that have been transcriptionally profiled, (2) genes that are annotated as putative regulatory factors that are absent from S. cerevisiae, (3) homologs of genes that are absent from S. cerevisiae and S. pombe and are of unknown biological function in N. crassa, and (4) apparent filamentous fungal-specific genes that do not have homologs in other organisms. The work outlined in this project will provide procedures, computational tools, and baseline transcriptional profiling data for wild type Neurospora grown under a variety of conditions and will pave the way for collaborative studies in the future. In addition to fostering similar studies in other filamentous fungi, these data will expedite the functional analysis of fungi in general including important human pathogens.